Automated sampling and reaction system for high pressure liquid chromatography and other types of detection

ABSTRACT

Automated sampling and reaction systems and methods of using the same are provided. The automated sampling and reaction system has a microreactor in fluidic communication with an external sampling valve. The external sampling valve is connected to a priming valve and can be configured to draw sample from a reactor or a reactor stream. The microreactor is connected to a reagent valve and an injection valve. The reagent valve can be configured to draw reagent from a reagent reservoir and discharge reagent to the microreactor to react with sample. The priming valve can be configured to draw wash from a wash reservoir and discharge wash to the external sampling valve to move sample from the external sampling valve to the microreactor. The injection valve is in fluidic communication with a column or detector and discharges the secondary sample into a solvent composition stream.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority to the U.S. ProvisionalApplication No. 62/100,252 filed Jan. 6, 2015 incorporated herein byreference.

BACKGROUND OF THE INVENTION

Most manufacturing industries use chromatography and other types ofseparation and detection systems to evaluate the process reactions ormanufacturing process lines. For example, pharmaceutical manufacturersoften use a chromatography system to monitor their process line bytaking samples at various times or at different points along the processline to ensure that a manufacturing batch is to specification. Samplesmay include complex mixtures of proteins, protein precursors, proteinfragments, reaction products, and other compounds, to list but a few.Other manufacturers may use their chromatography systems to profile acertain biochemical reaction, taking samples from the same point in theprocess line over time as the reaction progresses.

For every industry, preservation and transport of sample presents aparticular challenge as it is imperative that sample represents thebatch or flow stream that is being tested or otherwise investigated.During transport, changes in and to the physical and chemical makeup ofthe collected sample must be avoided for the measurements to bereliable. For example, the degradation of the sample due toenvironmental stress (i.e., heat, cold, oxygen) can cause an erroneousresult. Furthermore, any sample preparation prior to the measurementshould be performed with a minimum loss of sample and without unwantedmodifications and avoiding the additional of extraneous impurties.

The manner of acquiring samples for analysis can be manually intensive.Typically, an individual draws sample from a process line, reactor,reactor stream or the like. He or she then carries it to the separationand detection system and loads it into the system for injection andanalysis. Throughout this handling, care must be taken to label thesample properly and to ensure a well-documented chain of custody, orotherwise risk introducing uncertainty into the results.

If a sample needs to be converted to a form suitable for the measurementstep, sample preparations such as filtration, quenching, dilution, orderivatization are made before sample injection into a liquidchromatography system. In this case, the sample preparation apparatusmust be thoroughly washed to avoid contamination with the previoussample. Manual sample preparations can be wasteful and cost ineffectiveas larger amounts of the sample need to be taken. Manual samplepreparation introduces the risk of irreproducible results and affordssources of error to be generated during sample analysis.

For example, manual sample preparation for chemical derivatization priorto the injection is very time consuming. After manually acquiring thesample, the individual must next thoroughly mix the sample with areagent, or multiple reagents. Then, the individual must apply forexample heat to the container within which the derivatization occurs,ensuring that the heat is applied evenly throughout the container for aspecified time, sometimes hours, in order for the chemical reaction togo to completion. Each pre-analysis step can influence the overallaccuracy and reliability of the results. In some cases, variations inthe pre-analysis steps can introduce errors in the results that aregreater in magnitude than the properties of the sample that are beingmeasured.

SUMMARY OF THE INVENTION

Systems, methods and devices for automated sample preparation and samplereaction are provided herein. One aspect provides an automated samplingand reaction system including an external sampling valve, a microreactorin fluid communication with the external sampling valve, and aninjection valve connected to the microreactor. In exemplary embodiments,the external sampling valve can be connected to a priming valve. Theexternal sampling valve can be configured to draw sample from a reactoror a reactor stream. The priming valve can be configured to dischargewash to the external sampling valve. For example, the priming valve canbe configured to draw wash from a wash reservoir. The microreactor canbe connected to a reagent valve. For example, the reagent valve can beconfigured to draw reagent from a reagent reservoir or to dischargereagent into the microreactor. In some exemplary embodiments, thereagent valve can be configured to discharge reagent to the microreactorand the external sampling valve can be configured to discharge sample tothe microreactor to form a secondary sample. The microreactor, forexample, can be a chip, capillary, micro-structured or industrial typemicroreactor. In some embodiments, the injection valve can be configuredto discharge the secondary sample into a solvent composition stream. Inother aspects, a liquid chromatography system can include the automatedsampling and reaction system.

In some embodiments, the automated sampling and reaction system caninclude a mixing tee connected to the external sampling valve and themicroreactor. In further embodiments, a diluent valve can be connectedto the mixing tee. For example, the diluent valve can be configured todraw diluent from a diluent reservoir. Also, the diluent valve can beconfigured to discharge diluent to the mixing tee.

In a first configuration, the external sampling valve draws a discreteamount of sample or continuous sample from a reactor or reactor flowstream. In a second configuration, the external sampling valvedischarges drawn sample via a backwash discharged from the sample pumpto the mixing tee. In exemplary embodiments, the automated sampling andreaction system can operate under a pressure greater than about 1atmosphere or can be configured to draw from a non-pressurized source.When drawing from a non-pressurized source, the system can include atleast one external auxiliary sampling valve and at least one externalsample pump. For example, the external auxiliary sample valve can beconnected to the external sample pump, the external sampling valve andto the reactor or reactor stream. In some embodiments, the automatedsampling and reaction system can have a selection valve that isconnected to the external auxiliary sampling valve and to the externalsampling valve. The selection valve can be connected to a plurality ofthe external sampling valves.

In some embodiments, the automated sampling and reaction system caninclude a pumping system having one or more pumps working in combinationwith the priming valve, the diluent valve and/or the reagent valve. Forexample, the pumping system can have one or more pumps working incombination with the priming valve and the reagent valve. In exemplaryembodiments, the diluent pump can be configured to draw diluent from thediluent reservoir and discharge diluent to the mixing tee.

In another aspect, methods of quantitative analysis of a liquid solutionare provided. An exemplary embodiment of such methods includes the stepsof selecting a source of sample from a reactor or a reactor flow stream,acquiring sample from the reactor or the reactor flow stream through anexternal sampling valve, drawing wash through a priming valve in fluidiccommunication with the external sampling valve, reacting sample with areagent discharged from a reagent valve into a microreactor, dischargingthe secondary sample into a second sample loop of an injection valve,and injecting sample from the injection valve into a solvent compositionstream in fluidic communication with a column or detector. For example,the external sampling valve can be configured to draw sample into afirst sample loop. In exemplary embodiments, the microreactor can be influidic communication with the external sampling valve. The reagentvalve can be connected to the microreactor. In the microreactor, asecondary sample can be formed. In exemplary embodiments, the primingvalve can be configured to discharge wash from the second sample loop tothe external sampling valve.

In some embodiments, the sample can be acquired from the reactor or thereactor flow stream operating at pressure of more than one atmosphere.In some embodiments, sample can be acquired from the reactor or thereactor flow stream operating at a pressure of one atmosphere or less.For example, sample can be acquired by an external sample pump connectedto an external auxiliary sampling valve. The external pump thendischarges drawn sample via a backwash provided by a sample pump to amixing tee connected to the microreactor. In further embodiments, themethod can include the step of quenching sample at the mixing tee. Insome embodiments, the reagent can be an MS active, fluorescent rapidtagging reagent and the secondary sample can be an MS active,fluorescent biomolecule.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and further advantages of this invention may be betterunderstood by referring to the following description in conjunction withthe accompanying drawings, in which like numerals indicate likestructural elements and features in various figures. The drawings arenot necessarily to scale, emphasis instead being placed uponillustrating the principles of the invention.

FIG. 1 is a block diagram showing an automated sampling and reactionsystem utilizing the valve and pump assembly together with microreactorpresented herein.

FIG. 2A depicts the automated sampling and reaction system configured todraw sample from a pressurized sample source, to prime the sample pumpand to draw diluent and reagent into the diluent pump and reagent pump.

FIG. 2B depicts the automated sampling and reaction system configured tocollect sample from a non-pressurized sample source where the externalauxiliary valve is in the first configuration.

FIG. 2C depicts the automated sampling and reaction system configured tocollect sample from a non-pressurized sample source where the externalauxiliary valve is in the second configuration.

FIG. 2D shows the automated sampling and reaction system configured tocollect sample from a non-pressurized sample source where the externalsampling valve 22 is replaced with the external auxiliary sampling valve54 to draw sample and discharge sample from the third sample loop to themixing tee.

FIG. 2E shows the automated sampling and reaction system configured tocollect sample from a non-pressurized sample source where the externalsampling valve 22 is replaced with the external auxiliary sampling valve54 to load sample into the third sample loop.

FIG. 2F shows the automated sampling and reaction system configured tocollect sample from multiple non-pressurized sample sources.

FIG. 2G shows the automated sampling and reaction system configured tocollect sample from multiple pressurized sample sources.

FIG. 3 shows the automated sampling and reaction system configured todilute sample.

FIG. 4 shows the automated sampling and reaction system configured toderivatize sample.

FIG. 5 shows the automated sampling and reaction system configured toload sample into sample loop of the injection valve.

FIG. 6 shows the automated sampling and reaction system configured toinject sample into the solvent composition stream.

FIG. 7 provides a flow chart depicting the overall flow of the automatedsampling and reaction systems and methods of using the same that aredescribed herein.

DETAILED DESCRIPTION

Automated sampling and reaction systems for high pressure liquidchromatography or detectors for a wide variety of applicationsincluding, but not limited to, protein and peptide identification andquantitation, monitoring and analysis of cell culture media andnutritional content of food and feed are provided herein. The need tointerface synthetic organic reactions from either batch reactors orcontinuous flow reactors has been identified. The sample being analyzedcan be drawn from reactors of different sizes and can require dilutionto maintain solubility or decrease the amount of material beingintroduced into the chromatographic system to maintain a response fromthe detector. In other instances, the samples being withdrawn from theprocess may be environmentally unstable and any manual manipulation mayresult in the degradation of sample prior to analysis. In otherinstances, the reproducibility of a consistent sample being withdrawnindependent of the operator results in a more reproducible result.

Well-established chromatography technologies include High PerformanceLiquid Chromatography (“HPLC”), Ultra Performance Liquid Chromatography(“UPLC”) and Supercritical Fluid Chromatography (“SFC”). HPLC systemsuse high pressure, ranging traditionally between about 1,000 psi (poundsper square inch) to approximately 6,000 psi, to generate the flowrequired for liquid chromatography in packed columns. In contrast toHPLC, UPLC systems use columns with smaller particulate matter andhigher pressures approaching 20,000 psi to deliver the mobile phase. SFCsystems use highly compressible mobile phases, which typically employcarbon dioxide (CO2) as a principle component.

The automated sampling and reaction systems described herein can deliverturn-key analysis that can be optimized for high pressure liquidchromatography process and detection. The disclosed systems can be usedwith different types of detectors including mass spectrometry (“MS”),tunable ultraviolate/visible (“TUV”), photodiode array (“PDA”),evaporative light scattering, multi-angle light scattering, refractiveindex, conductivity, charged aerosol, chemiluminescent nitrogen detector(“CLNC”), electrochemical circular dischroism, polarimeter, nuclearmagnetic resonance (“NMR”), or fluorescent (“FLR”) detectors. Theautomated sampling and reaction systems are also useful forapplication-specific performance qualification, providing the sameresult: day-to-day, instrument-to-instrument, lab-to-lab around theworld.

The automated sampling and reaction system comprises an externalsampling valve, microreactor, priming valve, reagent valve, injectionvalve and a pumping system. The automated sampling and reaction systemmay further comprise a mixing tee and diluent valve. The pumping systemhas one or more pumps working in combination with the valves to draw anddischarge sample, diluent and/or reagent into the microreactor forsubsequent injection into a solvent composition stream. The pumpingsystem may include a sample pump, a diluent pump and/or a reagent pump.

More specifically, the external sampling valve is connected to thepriming valve, the mixing tee and a collection reservoir. The mixing teeis connected to the microreactor and the diluent valve. The microreactoris connected to the reagent valve and the injection valve. Sample reactsin the microreactor with reagent, diluent or other compounds to form asecondary sample which is then discharged into a solvent compositionstream flowing to the injection valve via a second sample loop. Thecombination of secondary sample and solvent composition stream is thendischarged to the liquid chromatography column or detector.

As described herein, the external sampling valve has two configurations.In a first configuration, the external sampling valve is configured todraw a discrete amount of sample or continuous sample from a reactor orreactor flow stream. In a second configuration, the external samplingvalve discharges drawn sample via wash discharged from the sample pumpto the mixing tee. Similarly, the reagent valve has two configurations.In a first configuration, the reagent valve draws reagent from a reagentreservoir. In a second configuration, the reagent valve dischargesreagent into the microreactor for reacting with sample. In addition, thepriming valve has two configurations. In a first configuration, wash isdrawn into the priming valve and in a second configuration, wash isdischarged from the priming valve into the external sampling valve.Likewise, the injection valve has two configurations. In a firstconfiguration, secondary sample is injected into the solvent compositionstream, or if secondary sample has not yet been loaded into the secondsample loop of the injection valve, a solvent composition stream flowsthrough the second sample loop. In the second configuration of theinjection valve, secondary sample flows through the second sample loopand solvent composition stream flows through the valve and out to acolumn or detector. Finally, when sample requires dilution, the diluentvalve is provided and has two configurations. In a first configuration,the diluent valve is configured to draw diluent from a diluentreservoir. In a second configuration, the diluent valve dischargesdiluent to the mixing tee.

These automated sampling and reaction systems couple the processanalysis system with novel flow reactor technology to enable continuous,online analysis of sample where sample preparation prior to analysis isrequired such as derivation, quenching and/or other types of samplepreparation. As described herein, triggering of an analysis draws samplefrom the reactor or reactor flow stream into the microreactor with orwithout prior dilution. The same trigger can launch the appropriatereagents to be pumped into microreactor.

As used herein, “online” means that the automated sampling and reactionsystem is connected directly to a process (or production) line toacquire samples from the process line in approximately real time withoutmanual intervention, then dilute, derivatize, load, and inject theacquired process samples for subsequent chromatographic analysis. Thechromatographic analysis thus occurs in parallel to the continuedoperation of the process line. No distinction is made here between aproduction line and a process line.

An “at-line” system means that the system is physically near butunconnected to the process line from which an individual acquires aprocess sample manually, carries and places the process sample into thesystem for processing.

An “in-line” system is one that is physically incorporated within theprocess line (i.e., the chromatographic analysis and process lineoperations in this instance are akin to serial processing), and can alsorefer to the direct real time detection process such as temperature,pressure or spectroscopy (such as raman and infrared) where the probesused are in direct contact with the process.

Advantageously, the systems and methods described herein increasereproducibility of the sample preparation and analysis, reducevariability due to handling, reduce the number of steps and manualmanipulations of the sample by the operator, reduce the overall time andeffort required to prepare the sample, improve throughput and decreasethe opportunity for imprecision and accuracy errors by the operator. Thepresent systems and methods do not require separate containers in orderto prepare a sample for injection to a detector or perform the dilution,quenching or derivatization. Contamination is avoided and smalleramounts of sample are needed for preparation and analysis.

Moreover, the automated sampling and reaction systems described hereinprovide online, synchronized addition of multiple reagents to samples atoptimal timing, concentration, temperature, and flow rate and withminimal interruption to sample preparation (i.e. allowing continuoussample preparation) prior to the injection into the column or detector.These systems and methods provide a controlled environment via the useof a microreactor in order to modify and/or convert sample by one ormore reagents. The amount of sample consumed is reduced and sampling isless disturbing to the process.

There are other advantages of the methods and systems disclosed herein.First, while sample volume must be sufficient to be transferred from thereactor to the injection valve, with the present systems and methods,sampling is largely undisturbed. These systems are designed to minimizesample diffusion. Using a back wash, sample travels in the middle of thetubular flow and, therefore, diffusion of sample at the edges of thetubing goes to waste and is not representative of the sample. The volumeof sample diffused is minimized and dispersion of the sample avoided.The sample largely remains intact. In addition, the amount of samplerequired for the detector is minimized because of the low rate ofdispersion of sample into the wash.

The Separation and Detection Systems

FIG. 1 is a block diagram of a separation and detection system 1 forseparating a mixture into its constituents as provided herein. Usefulseparation and detection systems 1 include liquid chromatographysystems, supercritical fluid chromatography systems, capillaryelectrophoresis, gas chromatography systems, massspectrometerslCP-MS/atomic adsorption source systems, fluorescencedetection, UV detection, UV-Visible spectroscopyisible, a cuvette, NMRtube or electromechanical. The separation and detection system 1 asdescribed herein includes an automated sampling and reaction system 2and a solvent delivery system 8. The automated sampling and reactionsystem 2 is in fluidic communication with a reactor 10 or a reactor flowstream (not shown). The automated sampling and reaction system 2 is alsoin fluidic communication with a solvent delivery system 8. The solventdelivery system 8 provides a solvent composition stream to the automatedsampling and reaction system 2. Subsequently, the solvent compositionstream is combined with sample and sent to, and received by, achromatographic column 6 or detector (not shown). The separation anddetection system 1 by way of the automated sampling and reaction system2 is directly connected to the reactor 10 or other vessel or reactorflow stream or other process line by tubing. The automated sampling andreaction system 2 can automatically or manually acquire samples from thereactor 10 or the reactor flow stream (not shown).

The automated sampling and reaction system 2 can acquire sample from apoint on a process flow stream, a reactor flow stream or directly fromthe reactor. The automated sampling and reaction system 2 can acquiresamples continuously or at different stages (location and/or time-based)of the manufacturing process. For example, the sample can be acquired atdifferent time intervals in order to derivatize sample during varyingstages of the process. In general, the reactor 10 or other vessel and/orthe reactor flow stream or other process lines are representative ofvarious process sources including manufacturing processes, beakerreactions, exit lines (cleaning validation), reaction chamber andfermentation reactions.

Importantly, the automated sampling and reaction system 2 allows forsample preparation to form a suitable secondary sample for measurementby the detector. Typically, sample preparation is an essential part ofchromatographic and spectroscopic analyses. The methods and systemsprovided herein provide a representative, reproducible and homogenoussolutions that are suitable for injection into the column forchromatographic analysis, or into an ICP-MS/atomic adsorption source orinto a cuvette or NMR tube for further characterization.

Using the systems and methods described herein, sample preparationprovides an aliquot that is relatively free of interferences and willnot damage the column or instrument and is also compatible with theanalytical method. In chromatography applications, sample can beinjected onto a chromatography column without affecting sample retentionor resolution or the stationary phase itself, and without interferingwith detection. Hence, it desirable to concentrate the analytes and/orderivatize them for improved detection or better separation. Inspectroscopy, samples should be free of particulates, compatible withspectroscopic sources and have appropriate viscosity to flow into anebulizer. In short, there are many types of sample preparationprotocols used in chromatography and spectroscopy and diverse methodsused. However, the systems and methods provided herein are able toaccommodate most protocols and methods for sample preparation.

Furthermore, the automated sampling and reaction system 2 describedherein can be located a substantial distance from the column 6 ordetector (not shown). As such, the term “remote” as used herein simplymeans separate (i.e. a separate module) or detached. The term remote isnot intended to mean that the automated sampling and reaction system 2is isolated from or otherwise positioned or located a significantdistance away from the column 6 or the detector (not shown), and/or thereactor 10 or reactor flow stream. Hence, the devices and methodsdescribed herein may include those situations where the automatedsampling and reaction system 2 is close to or in proximity to the column6 or the detector, and to the reactor 10 or reactor flow stream.

The solvent delivery system 8 can include a low-pressure pumping system(not shown) in fluidic communication with reservoirs 4 from which thepumping system draws liquid solvents through tubing (not shown). In alow-pressure pumping system, the mixing of solvents typically occursbefore the pump (not shown). The solvent delivery system 8 also may havea mixer (not shown) in fluidic communication with the solvent reservoirs4 to receive various solvents in metered proportions. This mixing ofsolvents occurs in accordance with an intake profile, and produces asolvent (mobile phase) composition that remains unchanged (isocratic) orvaries over time (gradient). Hence, the pumping system of a solventdelivery system 8 is in fluidic communication with a mixer and can drawa continuous flow of solvent mixture therefrom for delivery to theliquid chromatography column 6 or detector (not shown). To draw anddeliver the solvent mixture, the pumping system can, for example,provide a flow rate in the range of about 0.010 ml/min to about 2 ml/minat about 15,000 psi. On the other hand, the flow rate can be a “nanoflow” (or down to 200 nL/min) or an HPLC flow (analytical 10 mL/min andpreparative to 20-50 mL/min) having pressures from 1000 psi to 20,000psi. Examples of pumping systems that can be used to implement thepumping system include, but are not limited to, the ACQUITY HPLC BinarySolvent Manager, manufactured by Waters Corp. of Milford, Mass. See,e.g., US 2012/0303167 at ¶ [0019]. Other useful examples of pumpingsystems include, but are not limited to, single piston pump solventdelivery systems, dual piston reciprocating pump systems, alternativebinary pumps, quaternary pump solvent delivery systems, and ternary pumpsystems.

Hence, by way of example, the solvent delivery system 8 can be a binarysolvent manager (“BSM”), which uses two individual serial flow pumps todraw solvents from reservoirs 4 and deliver the solvent compositionstream to the automated sampling and delivery system 2. Here, each ofthe BSM's two independent pumps contains two linear-drive actuators.Each actuator pair comprises a single reciprocating serial pump thatdelivers precise flow of a single solvent. The two pump systems combinetheir two solvents 18 at a filter/tee mixer. From there, the solventmixture flows into the automated sampling and reaction system 2. Agradient elution program is commonly used so that the eluent composition(and strength) is steadily changed during the analysis. This increasesseparation efficiency, decreases the retention time and improves peakshape by minimizing tailing. See, e.g., T Jiang Y, Viadya L, The WatersACQUITY® Ultra-Performance Liquid Chromatograph and the Micromass QuatroPremier Triple Quadrupole Mass Spectrometer, December, 2012.

The separation and detection system 1 may also include a data system 100that is in signal communication with the automated sampling and reactionsystem 2 and the solvent delivery system 8. The data system 100 has aprocessor and a switch (not shown), e.g., an Ethernet switch forhandling signal communication between the solvent delivery system 8 andthe automated sampling and reaction system 2. In addition, the datasystem 100 is programmed to implement the various phases of operationperformed by the automated sampling and reaction system 2 (e.g. turningpumps on and off, rotating valves) in order to automatically acquire,dilute, quench and/or derivatize a process sample and introduce thetreated process sample to the solvent composition stream, as describedherein. Furthermore, a host computing system 102 is in communicationwith the data system 100, by which the user can download variousparameters and profiles to affect the data system's performance.

The Automated Sampling and Reaction System

The automated sampling and reaction system described herein can samplefrom concentrated reactions and dilute sample over a wide range. Forexample, sample dilution can range from about 1 to 99 units of diluentto 1 unit of sample. However, dilution range of sample can extend toabout 5000 to 1, depending on the accuracy of the remote pump. Theautomated sampling and reaction system also allow sample quenching, ifneeded. In these systems, sample is prevented from contacting pumpswhich increases pump life due to the lack of harsh conditions andprevents contamination. The automated sampling and reaction system 2 canprocess sample volumes of about 100 μl.

As shown in the figures, the separation and detection system 1 includesautomated sampling and reaction system 2, a solvent delivery system 8and a column 6 or a detector. The automated sampling and reaction systemfurther includes an external sampling valve 22, a priming valve 24, adiluent valve 26, a reagent valve 28 and an injection valve 30. Inaddition, the automated sampling and reaction system 2 further includesa sample pump 32, a diluent pump 34 and a reagent pump 36, a mixing tee18 and a microreactor 12.

Each of the valves is a separate, independently operable rotary valvehaving a plurality of fluidic ports and one or more flow-throughconduits. Although described primary as rotary valves, any one or moreof these valves: priming, sampling, process-selection, and/or injection,can be other types of valve including, but not limited to, slidervalves, solenoids, and pin valves. Each flow-through conduit provides apathway between a pair of neighboring fluidic ports. When a given valverotates, its flow-through conduits move clockwise or counterclockwise,depending upon the valve's direction of rotation. This movement operatesto switch the flow-through conduit to a different of neighboring fluidicports, establishing a fluidic pathway between that different pair whileremoving the pathway from the previously connected pair of fluidicports.

Further, the valves are sometimes described herein with respect to aparticular configuration and rotation thereof, especially as it mayrelate to the processing of sample within the automated sampling andreaction system 2. However, the valves, including the external samplingvalve 22, the priming valve 24, the diluent valve 26, the reagent valve28 and the injection valve 30, could each rotate in an oppositedirection from that which is described herein and shown in the figures(i.e. clockwise as opposed to counterclockwise or counterclockwise asopposed to clockwise) and still accommodate the same functionality andoverall workings of the automated sampling and dilution system 2provided herein. In short, the valves and the operation of the valvesare not limited to the manner of rotation or a specific configurationdescribed herein.

In addition, unless otherwise specified, all connections are fluidic andprovide for fluid flow, including but not limited to, tubing connectionsbetween fluidic ports and devices such as the reactor, the reactor flowstream, valves, pumps, reservoirs and other apparatus that are describedherein. Such connections are typically made via tubing ranging in sizefrom 0.005 to 0.150 inches and made of stainless steel, PEEK, Teflon,and/or any material suitable for the pressure and composition of thesample. Also, flow-through conduits are fluidical connections where theports and conduits are fluidically connected to each other and/or otherdevices described. Hence, when it is stated that a device, fluidic portor flow-through conduit is connected or in fluidic communication withthe other, this means and should be understood to mean that suchconnection is fluidic unless otherwise noted.

The external sampling valve 22 has a first sample loop 40, six fluidicports 22-1, 22-2, 22-3, 22-4, 22-5 and 22-6 and three flow-throughconduits 22-11, 22-12 and 22-13. The first sample loop 40 connectsfluidic ports 22-1 and 22-4. Tubing connects fluidic port 22-2 to thereactor 10. Tubing connects fluidic port 22-3 to a collection reservoir44. Tubing connects fluidic port 22-5 to fluidic port 24-1 of thepriming valve 24. Further, tubing connects fluidic port 22-6 to themixing tee 18. As shown in FIG. 2, in the idle configuration, samplepump 32, diluent pump 34 and reagent pump 36 are off and not running.However, sample could flow into fluidic port 22-1 into the externalsampling valve 22 through flow-through conduit 22-11 and out fluidicport 22-1 into the first sample loop 40 even when not sampling. Ifreactor or process flow stream operates under pressure, sample may flowout of the first sample loop 40 into fluidic port 22-4 through flowthrough conduit 22-12 out fluidic port 22-3 and into the collectionreservoir 44.

Similarly, the injection valve 30 has a second sample loop 42, sixfluidic ports 30-1, 30-2, 30-3, 30-4, 30-5 and 30-6 and threeflow-through conduits 30-11, 30-12 and 30-13. The second sample loop 42connects fluidic ports 30-1 and 30-4. Tubing connects fluidic port 30-2to a waste reservoir 38. Tubing also connects fluidic port 30-3 to themicroreactor 12. Further, tubing connects the solvent delivery system 8to fluidic port 30-5. Also, tubing connects fluidic port 30-6 to thecolumn 6 or a detector (not shown). Flow-through conduit 30-11 providesa fluidic pathway between fluidic port 30-1 and fluidic port 30-6.Similarly, flow-through conduit 30-13 provides a fluidic pathway betweenfluidic port 30-4 and fluidic port 30-5. The injection valve 30 has thecapacity to handle volumes of 10 micoliters to 100 microliters and canbe scaled.

During operation, the solvent delivery system 8 should be on in order tomaintain minimal disturbance to the solvent composition stream and toprovide a solvent composition stream having a fluidic pathway intofluidic port 30-5 through flow-through conduit 30-13 out fluidic port30-4 through the second sample loop 42. This solvent composition streamfluidic pathway can continue into fluidic port 30-1 through flow-throughconduit 30-11 and out fluidic port 30-6 to the column 6 or detector (notshown).

As shown in the figures, the priming valve 24 has seven fluidic ports24-1, 24-2, 24-3, 24-4, 24-5, 24-6 and 24-7 and one flow-through conduit24-11. Tubing connects fluidic port 24-6 to a wash reservoir 46. Tubingalso connects fluidic port 24-7 to the sample pump 32. Tubing furtherconnects fluidic port 24-1 to fluidic port 22-5 of the external samplingvalve 22.

Similarly, the diluent valve 26 has seven fluidic ports 26-1, 26-2,26-3, 26-4, 26-5, 26-6 and 26-7 and one flow-through conduit 26-11.Tubing connects fluidic port 26-6 to a diluent reservoir 48 and fluidicport 26-7 to the diluent pump 34. Tubing further connects fluidic port26-1 to the mixing tee 18.

The reagent valve 28 also has seven fluidic ports 28-1, 28-2, 28-3,28-4, 28-5, 28-6 and 28-7 and one flow-through conduit 28-11. Tubingconnects fluidic port 28-6 to a reagent reservoir 50 and fluidic port28-7 to the reagent pump 36. Tubing also connects fluidic port 28-1 tothe microreactor 12.

The sample pump 32, the diluent pump 34 and the reagent pump 36 are eachpositive displacement pumps. During startup, a liquid positivedisplacement pump cannot simply draw air until the feed line and pumpfill with the liquid that requires pumping. Typically, an operator mustintroduce liquid into the system to initiate the pumping. While loss ofprime is usually due to ingestion of air into the pump, the clearancesand displacement ratios in pumps for liquids and other more viscousfluids usually cannot displace air due to its lower compressibility. Inthe present assembly, however, the priming valve 24, the diluent valve26 and the reagent valve 28 replace the need for manually introducingliquid into the sample pump 32, the diluent pump 34 and the reagent pump36.

The automated sampling and reaction system 2 can be used to monitor anyprocess or reaction where the reactor or the reactor flow stream is nearor far away. The size and length of the tubing required can bemathematically represented as follows:

Δp=8*ρ*(V ²)/(π² *D ⁴)*κ*L/D*0.00014504, where

ρ=solvent density (kg/m³)

V=flow velocity (m³/s)

D=tube diameter (m)

λ=Coefficient of friction

L=length of tube

0.00014504=kPa to psi

Sample can be drawn from a reactor 10 or reactor stream operating underpressure or for a non-pressurized reaction where the reactor or othervessel is not operating under pressure (greater than about 1 atmosphereor 14.7 psi at sea level). The automated sampling and reaction system 2described herein dilutes sample at the mixing tee 18. However, thissystem 2 can work without sample dilution. Likewise, reagent can bereacted with diluted or un-diluted sample in the microreactor 12 priorto injection of the sample into the column 6 or detector. However, theautomated sampling and reaction system 2 can simply provide a directsample load to the injection valve 30 without sample dilution oraddition of reagent.

Sample volume must be large enough to be transferred from the reactor 10to the injection valve 30. In the present system 2, sample flow islargely undisturbed and unaffected by the system 2. However, if thesample is first diluted, a larger volume is created and sample can betransferred farther before unacceptable levels of diffusion are reached.For example, although the ends of a sample band undergo diffusion, themiddle portion of the sample band away from the ends will remainunaffected. A diluted sample will have a larger volume of sample awayfrom the ends that is unaffected by diffusion in comparison to anundiluted sample. A diluted sample can therefore be moved farther thanan undiluted sample. Furthermore, because tubing diameters are narrow,sample diffusion is minimized regardless of distance transferred. In thepresent systems, samples largely remain intact because contact areabetween sample and solvent is minimized. For example, the diameter ofthe tubing can be small, e.g., as small as approximately 4 mil (about100 μm.) Diffusion is a concentration-driven mass transfer process thatcan be defined as the mass transferred per unit area per unit time.Small diameter tubing provides a corresponding small area over whichdiffusion can occur, thereby reducing diffusion. With the use of abackwash in the system 2, dispersion of the sample is avoided. Inaddition, the amount of sample required for the column 6 or the detector(not shown) is minimized because of the low rate of dispersion of sampleinto the wash.

As shown in figures and described in more detail below, in operation,the solvent delivery system is turned on. Likewise, the sample pump 32,the diluent pump 34 and the reagent pump 36 are turned on and charged(FIG. 2). The sample pump 32 draws wash from the wash reservoir 46through the priming valve 24 through fluidic port 24-6 into flow-throughconduit 24-11 and out fluidic port 24-7. Sample is then drawn from thereactor 10, or other sample source, by the sample pump 32 into theexternal sampling valve 22 at fluidic port 22-2 creating a fluidicpathway of sample through flow-through conduit 22-11 and out fluidicport 22-1 into the first sample loop 40, and into fluidic port 22-4through flow-through conduit 22-12 and out fluidic port 22-12 into thecollection reservoir 44 (FIG. 3).

As shown in FIG. 3, to mix sample and dilute, the priming valve 24 andthe external sampling valve 22 are rotated so that sample pump 32discharges wash from the priming valve 24 to the external sampling valve22 in order to backwash sample transferring to the mixing tee 18. At themixing tee 18, sample can be mixed with diluent or can simply flowthrough the mixing tee to the microreactor.

As shown in FIG. 4, additional sample preparation can occur in themicroreactor 12 where sample reacts with a reagent or other reactantwhich is discharged by the reagent pump 36 through the reagent valve 28.Sample is then sent to the injection valve 30, loaded into the secondsample loop 42 and injected into the solvent composition stream pumpedfrom the solvent delivery system 8 (FIG. 5). The sample in the solventcomposition stream is pushed to the column 6 or a detector (FIG. 6).During this step, the pumps 32, 34 and 36 can be charged to be ready fornext injection.

Sample Collection

As noted above, the priming valve 24 is connected to the sample pump 32at fluidic port 24-7. The priming valve 24 is also connected to the washreservoir 46 at fluidic port 24-6. The diluent valve 26 is connected tothe diluent pump 34 at fluidic port 26-7 and to the diluent reservoir 48at fluidic port 28-6. The reagent valve 28 is connected to the reagentreservoir 50 at fluidic port 28-6 and to the reagent valve 36 at fluidicport 28-7.

FIG. 2A depicts the automated sampling and reaction system 2 configuredto draw sample from the reactor 10 and to draw wash from the washreservoir 46 into the priming valve 24. For sample collection, thesample pump 32 is turned on and charged. If sample is to be diluted, thediluent pump 34 is also preferably turned on and charged. The reagentpump 36 can also be turned on and charged or can remain off. The samplepump 32, the wash pump 34 and the reagent pump 38 can be turned on andcharged in parallel or sequentially.

In addition, during sample collection, the solvent delivery systemdischarges a solvent composition stream into the injection valve 30 atfluidic port 30-5, where a fluidic pathway is provided from fluidic port30-5 through flow-through conduit 30-13 into fluidic port 30-4 andthrough the sample loop 42 into fluidic port 30-1 through flow-throughconduit 30-11 and out fluidic port 30-6 to the column 6 or otherdetector.

For sample collection, as shown in FIG. 2A, the priming valve 24 isconfigured to provide a fluidic pathway between the sample pump 32 andwash reservoir so that wash is drawn from the wash reservoir 46 into thepriming valve 24 at fluidic port 24-6 through the flow-through conduit24-11 and out fluidic port 24-7 to the sample pump 32. Likewise, in itsfirst configuration the external sampling valve 22 is configured toprovide a fluidic pathway between the reactor 10 or other source ofsample to the collection reservoir 44 so that sample is drawn ordischarged from the reactor 10 through fluidic port 22-2 throughflow-through conduit 22-11 and out fluidic port 22-1 through the firstsample loop 40 into fluidic port 22-4 through flow-through conduit 22-12and out fluidic port 22-3 to the collection reservoir 44. Recyclingsystems and devices can be connected to the collection reservoir 44 ordirectly to the external sampling valve 22 to allow for the sample toflow back to the reactor, with or without further treatment orprocessing of the sample.

As also shown in FIG. 2A, the reagent valve 28 can be configured toprovide a fluidic pathway between the reagent reservoir 50 and thereagent pump 36 such that the reagent pump 36 draws reagent from reagentreservoir 50. Optionally, the diluent valve 26 can be configured toprovide a fluidic pathway between the diluent pump 34 and the diluentreservoir 48. Specifically, a fluidic pathway is provided from thediluent reservoir 48 through the diluent valve 26 at fluidic port 26-6through flow-through conduit 26-11 and out fluidic port 26-7. As anoption, at the sample collection step, the reagent pump 36 draws reagentfrom the reagent reservoir 50 to the reagent pump 28 through the reagentvalve 28 as the flow-through conduit 28-11 provides fluidic pathwaybetween fluidic port 28-6 and fluidic port 28-7. As an option, reagentcan be contained in, and drawn from, the diluent reservoir 48 by thediluent pump 34 and therefore, a fluidic pathway of reagent is providedfrom the diluent reservoir 48 through the diluent valve 26 at fluidicport 26-6 through flow-through conduit 26-11 and out fluidic port 26-7.

Automated Sampling and Reaction System Sampling from a Non-PressurizedSource

As shown in FIG. 2B, FIG. 2C and FIG. 2F, the automated sampling andreaction system 2 can include one or more external auxiliary samplingvalves 54 and one or more external sample pumps 52 combined in a waythat allows sampling from one or more non-pressurized supply of samplesuch as a reactor or reactor stream 10 or dissolution bath (not shown).The automated sampling and reaction system 2 can be used to draw aplurality of process samples from a single source of sample or, with theuse of a selection valve 56, a plurality (more than one) of reactors 10or dissolution baths or other sources of sample.

As shown in the figures, the external auxiliary sampling valve 54 hasten fluidic ports 54-1, 54-2, 54-3, 54-4, 54-5, 54-6, 54-7, 54-8, 54-9,and 54-10 and five flow-through conduits 54-11, 54-12, 54-13, 54-14, and54-15. Fluidic ports 54-1 and 54-8 are plugged and not used for fluidflow of sample. A third sample loop 55 connects fluidic ports 54-3 and54-6. Tubing connects the external auxiliary sampling valve 54 to thereactor 10 and to the external sample pump 52. Tubing also connects theexternal auxiliary sampling valve 54 to the external sampling valve 22.More specifically, tubing connects fluidic port 54-10 to thenon-pressurized reactor 10. Tubing further connects fluidic ports 54-7and 54-9 to the external sample pump 52. In addition, tubing connectsthe fluidic port 54-2 to the fluidic port 22-2 of the external samplingvalve 22.

The external sample pump 52 is a positive displacement pump. Duringstartup, a liquid positive displacement pump cannot simply draw airuntil the feed line and pump fill with the liquid that requires pumping.Typically, an operator must introduce liquid into the system to initiatethe pumping. While loss of prime is usually due to ingestion of air intothe pump, the clearances and displacement ratios in pumps for liquidsand other more viscous fluids usually cannot displace air due to itshigher compressibility.

The selection valve 56 has seven fluidic ports 56-1, 56-2, 56-3, 56-4,56-5, 56-6 and 56-7 and one flow-through conduit 56-11. The number ofselection valves 56 depends, in part, on the number of externalauxiliary sampling valves 54. It is optional for the automated samplingand reaction system 2 to include one or more selection valves 56 withonly one external auxiliary sampling valve 54. However, the automatedsampling and reaction system 2 having a plurality of external auxiliarysampling valves 54 requires one or more selection valves 56.

When samples are drawn from a plurality of reactors 10, there is atleast one external auxiliary sampling valve 54 in fluidic communicationwith each reactor 10. Furthermore, when the automated sampling andreaction system 2 has two or more external auxiliary sampling valves 54,at least one selection valve 56 is required. In other words, while aplurality of external auxiliary sampling valves 54 and/or a plurality ofselection valves 56 could be connected to a single reactor 10, at leastone selection valve 56 must be provided for the automated sampling andreaction system 2 having two or more external auxiliary sampling valves54. For the automated sampling and reaction system 2 having a pluralityof external auxiliary sampling valves 54, tubing connects fluidic port54-2 of each external auxiliary sampling valve 54 to the selection valve56. Fluidic port 54-2 can be connected to any of fluidic ports 56-1,56-2, 56-3, 56-4, 56-5 or 56-6 of the selection valve 56 and inalternative combinations.

More specifically, as shown in FIGS. 2B, 2C and 2F, tubing connectsfluidic ports 54-7 and 54-9 of the external auxiliary sampling valve 54to the external pump 52. Tubing connects fluidic port 54-10 of theexternal auxiliary sampling valve 54 to the reactor 10 or other sourceof sample. Tubing connects fluidic ports 54-2 of the external auxiliarysampling valve 54 to fluidic port 22-2 of the external sampling valve 22when a single external auxiliary sampling valve 54 is used (FIGS. 2B and2C). Where a plurality of external auxiliary valves 54 are used, tubingcan connect fluidic port 54-2 of the external auxiliary valve 54 to eachof fluidic port 56-1, 56-2, 56-3, 56-4, 56-5 or 56-6 of the selectionvalve 56 (FIG. 2F).

FIG. 2F depicts an example of the automated sampling and reaction system2 having three external auxiliary sampling valves 54 and one selectionvalves 56. Fluidic port 56-7 of each selection valve 56 is connected tothe external sampling valve 22. Fluidic port 54-2 of each externalauxiliary sampling valve 54 is connected to the selection valve 56 atfluidic ports 56-1, 56-2 and 56-3. As shown, fluidic port 56-7 of theselection valves 56 is connected to fluidic port 22-2 of the externalsampling valve 22.

The various combinations of valve configurations for the selection valve56 and the external auxiliary sampling valves 54 effectively determinesthe fluidic pathway from the reactor 10, the external auxiliary samplingvalve 54, and the selection valve 56 to the external sampling valve 22.In short, the configuration of the selection valve 56 determines thefluidic pathway of sample from which reactor 10 to the external samplingvalve 22. Clockwise and counterclockwise rotation of the externalauxiliary sampling valve 54 achieves the same configuration. Optionally,the selection valve 56 can have eight fluidic ports with a flow-throughconduit (not shown).

Sample Collection from a Single Non-Pressurized Sample Source

As described immediately above, the automated sampling and reactionsystem 2 can be configured to draw a sample from a non-pressurizedsample source. As described herein, the external auxiliary samplingvalve 54 can toggle between two configurations, the first configurationand the second configuration, to perform three steps: draw sample, loadsample into the third sample loop 55 and discharge sample. In the firstconfiguration shown in FIG. 2B, the external sample pump 52 draws samplefrom the reactor 10. In the second configuration shown in FIG. 2C, theexternal sample pump 52 discharges sample into the third sample loop 55of the external auxiliary sampling valve 55. Returning to the firstconfiguration of the external auxiliary sampling valve 54, the externalsample pump 52 discharges sample from the third sample loop 55 to theexternal sampling valve 22.

More specifically, as shown in FIG. 2B, in the first configuration, theexternal sample pump 52 draws sample from the reactor 10 through fluidicport 54-10 of the external auxiliary sampling valve 54 into flow-throughconduit 54-15 and out fluidic port 54-9 to the external sample pump 52.As shown in FIG. 2C, in the second configuration, the external auxiliarysampling valve 54 has been rotated one port position clockwise orcounterclockwise. In this second configuration, the external sample pump52 discharges sample into fluidic port 54-7 of the external auxiliarysampling valve 54 through flow-through conduit 54-14 and out fluidicport 54-6 through the third sample loop 55 and then into fluidic port54-3 through flow-through conduit 54-12 and out fluidic port 54-2 to thefluidic port 22-2 of the external sampling valve 22 and continuingthrough fluidic conduit 22-11 and out fluidic port 22-1 to load sampleinto the first sample loop 40 of the external sampling valve 22.

The external auxiliary sampling valve 54 then rotates counterclockwisetoggling back to the first configuration. The external sample pump 52displaces sample drawn through the third sampling loop 55. The samplepump 32 discharges wash to external sampling valve 22 as describedimmediately below in FIGS. 3 through 7. If the configuration of theexternal sampling valve 22 is not changed and remains as described inFIG. 2, the sample will be discharged into the collection reservoir 44,provided the external sample pump 52 is turned on.

Alternatively, the external sampling valve 22 may be replaced with theexternal auxiliary sampling valve 54, as shown in FIGS. 2D and 2E. Here,the fluidic ports 54-2 and 54-5 of the external auxiliary sampling valve54 are plugged and not used for sample flow. Fluidic ports 54-1 and 54-8are not plugged. As shown in FIGS. 2D and 2F, tubing connects fluidicports 54-4 and 54-6 of the external auxiliary sampling valve 54 to theexternal pump 52. Tubing connects fluidic ports 54-1 and 54-3 of theexternal auxiliary sampling valve 54 to the reactor 10 or other sourceof sample. Tubing connects fluidic port 54-8 of the external auxiliarysampling valve 54 to fluidic port 24-1 of the priming valve 24. Tubingfurther connects fluidic port 54-9 of the external auxiliary samplingvalve 54 to the mixing tee 18.

FIGS. 2D and 2E each show the first and the second configurations ofexternal auxiliary sampling valve 54, respectively. As shown in FIG. 2D,in the first configuration, sample is drawn from the reactor 10 throughfluidic port 54-3, into flow-through conduit 54-12 and out fluidic port54-4 to the external sample pump 52. As shown in FIG. 2E, in the secondconfiguration, the external sample pump 52 discharges sample intofluidic port 54-6 of the external auxiliary valve 54 throughflow-through conduit 54-14 and out fluidic port 54-7 filling the thirdsample loop 55 into fluidic port 54-10 through flow-through conduit54-11 and out fluidic port 54-1 recirculating back to the reactor 10.Sample constantly flows through the third sample loop 55 in this manner.The external auxiliary sampling valve 54 then rotates counterclockwisetoggling back to the first configuration.

Sample Collection from Multiple Non-Pressurized Sample Sources

Samples can be taken from one or more non-pressurized sources (reactors,reactor flow streams, and the like) sequentially or simultaneously, inseries or in parallel. Each external auxiliary sampling valve 54 drawssample independently from the other. However, the number of samples thatcan be taken depends on the number of external auxiliary sampling valves54 provided in the automated sampling and reaction system 2. Also, foreach external auxiliary sampling valve 54, an external sample pump 52 isprovided. Further, if a plurality of external auxiliary sampling valves54 is required, there must be one selection valve 56 and there can be upto six external auxiliary sampling valves 54 for every one selectionvalve 56.

To draw sample from multiple reactors 10 or other sources, the externalsample pump 52 must be on. As described above, the external auxiliarysampling valves 54 alternate between two configurations and do so, inthree steps. Each of the external auxiliary sampling valves 54 can be inthe same configuration or can be in the other configuration, i.e., thefirst configuration versus the second configuration described above.

By way of example, FIG. 2F shows automated sampling and reaction system2 having three external auxiliary sampling valves 54 and one selectionvalve 56. As shown in FIG. 2F, the external auxiliary sampling valves 54can be in different configurations. In this example, sample can be drawnfrom the reactor 10 through fluidic port 54-10 into flow-through conduit54-15 and out fluidic port 54-9 of the external auxiliary sampling valve54. Concurrently, sample could be displaced in the third sample loop 55of another external auxiliary sampling valve 54 and discharged toselection valve 56.

Sample Collection from Multiple Pressurized Sample Sources

The automated sampling and reaction system 2 can be configured to drawsamples from more than one source of sample under pressure, in sequenceor in parallel. As shown in FIG. 2G, to draw sample from multiple samplesources under pressure, the automated sampling and reaction system 2 caninclude a plurality of external sampling valves 22 and at least oneselection valves 56. The external sampling valves 22 and selectionvalve(s) 56 are combined in a way that allows sampling from one or morepressurized supply of sample such as a reactor 10 or reactor stream ordissolution bath (not shown), or other sources of sample. The number ofselection valves 56 depends, in part, on the number of the externalsampling valves 22. However, at least one selection valve 56 must bepresent when five or less external sampling valves 22 are used.

FIG. 2G depicts an example of the automated sampling and reaction system2 comprising three external sampling valves 22 and one selection valve56. As described herein, each external sampling valve 22 has six fluidicports 22-1, 22-2, 22-3, 22-4, 22-5 and 22-6 and three flow-throughconduits 22-11, 22-12 and 22-13. The first sample loop 40 connectsfluidic ports 22-1 and 22-4. As shown, tubing connects fluidic port 22-2of each of the external sampling valves 22 to the reactor 10, andfluidic port 22-3 to the collection reservoir 44. Tubing furtherconnects fluidic port 22-5 of each external sampling valve 22 to fluidicports 24-1, 24-2 and 24-3 of the priming valve 24 and fluidic port 22-6of each external sampling valve 22 to fluidic ports 56-1, 56-2 and 56-3of the selection valve 56. Fluidic port 22-5 can be connected to any ofthe fluidic ports 24-1, 24-2, 24-3, 24-5 or 24-6 of the priming valve 24and in alternative combinations. However, at least one fluidic port ofthe priming valve 24 must be connected to the wash reservoir 46. Fluidicport 24-7 is then connected to the sample pump 32. Similarly, fluidicport 22-6 can be connected to any of the fluidic ports 56-1, 56-2, 56-3,56-4, 56-5 or 56-6 and in alternative combinations. Tubing also connectsfluidic port 56-7 to the mixing tee 18.

The combination of the first and second configurations of the externalsampling valve, together with the configurations of the selection valve56 and the priming valve 24, effectively determine the fluidic pathwayfrom the reactor 10 to the mixing tee 18. For example, as shown in FIG.2G, two external sampling valves 22 are shown in the first configurationproviding a fluidic pathway between the reactor 10 and the collectionreservoir 44. The other external sampling valve 22 is in the secondconfiguration, providing a fluidic pathway between the reactor 10 andthe mixing tee 18.

Sample Dilution

FIG. 3 shows the automated sampling and reaction system 2 configured todilute (or quench) sample at the mixing tee 18. As shown, theconfigurations of the priming valve 24, the external sampling valve 22and the diluent valve 26 are changed from those shown in FIG. 2 so thatsample and diluent are discharged into the mixing tee 18.

Specifically, in its second configuration, the priming valve 24 isrotated counterclockwise by one port position such that the flow-throughconduit 24-11 connects fluidic port 24-7 to fluidic port 24-1 toestablish a fluidic pathway between the sample pump 32, the primingvalve 24 and the external sampling valve 22. Likewise, in its secondconfiguration, the external sampling valve 22 is rotated clockwise byone port position such that the flow-through conduit 22-11 connectsfluidic port 22-1 to fluidic port 22-6, establishing a fluidic pathwaybetween the first sample loop 40 and the mixing tee 18. In addition, theflow-through conduit 22-13 connects fluidic port 22-5 to fluidic port22-4 such that a fluidic pathway is established between the primingvalve 24 and the mixing tee 18. Counterclockwise rotation of theexternal sampling valve 22 by one port position achieves the sameconfiguration. Also, in its second configuration, the diluent valve 26is rotated counterclockwise one port position such that flow-throughconduit 26-11 connects fluidic port 26-1 to fluidic port 26-7establishing a fluidic pathway between the diluent pump 34 and themixing tee 18.

In short, for sample dilution, sample is discharged from the firstsample loop 40 of the external sampling valve 22 through fluidic port22-1 into flow-through conduit 22-11 and out fluidic port 22-6 to themixing tee 18. Simultaneously, the dilute pump 34 discharges diluentfrom the diluent valve 26 through fluidic port 26-7 into flow-throughconduit 26-11 and out fluidic port 26-1 to the mixing tee 18. Sample ismixed with diluent and diluted at the mixing tee 18. For sampledilution, the sample pump 32 and the diluent pump 34 must dischargesample and diluent concurrently to the mixing tee 18. The flow rates ofthe pumps 32 and 34 determine the dilution ratio (overall dilution flowrate to process sample flow rate). Consider, for example, an overalldilution flow rate of 100 μl/min, with the sample pump 32 discharging 10μl/min of sample while the diluent pump 34 discharges 90 μl/min ofdiluent: the result is a 10:1 dilution. When, for example, the samplepump 32 pushes 50 μl/min, while the diluent pump 34 pushes 50 μl/min,the result is a 2:1 dilution. Furthermore, the timing of the system issuch if sample is retained in the mixing tee 18 too long, sample may getdiffused. So, sample should be diluted as soon as possible. Moreover, bydiluting sample as it is moved through the mixing tee 18, a largervolume is created and the sample can be moved greater distances.

Before or during sample dilution, the configuration of the reagent valve28 can change to a second configuration, by rotating counterclockwise byone port position such that the flow-through conduit 28-11 connectsfluidic port 28-1 to fluidic port 28-7. In this configuration, thereagent pump 36 discharges reagent through fluidic port 28-7 throughflow-through conduit 28-11 and out fluidic port 28-1 to the microreactor12. However, the reagent valve 28 does not have to change at the sametime that the configurations of the external sampling valve 24 and thepriming valve 26 or before the sample preparation step.

As noted above, sample can be drawn from a reactor or reactor flowstream operating under pressure or under non-pressurized reaction wherethe reactor or other vessel is not operating under pressure (i.e., at apressure greater than about 1 atmosphere or 14.7 psi at sea level). Thesystem 2 described herein can dilute a sample drawn from the reactor bydiluting sample at the mixing tee 18 (also sometimes referred to asdirect dilution line load). Alternatively, the system 2 can provide adirect sample load to the injection valve 30 via the microreactor 12without sample dilution.

Sample Preparation

FIG. 4 depicts the automated sampling and reaction flow system 2 havingvalves configured to add reagent or other reactants to sample in themicroreactor 12. The configurations of the priming valve 24, theexternal sampling valve 22 and the diluent valve 26 remain asimmediately described above in the dilution step and as shown in FIG. 3.At this time, the reagent valve 28 is configured in a secondconfiguration such that flow-through conduit 28-11 connects fluidic port28-1 to fluidic port 28-7 and provides a fluidic pathway between thereagent pump 36 and the microreactor 12. Reagent pump 26 dischargesreagent and/or other reactants into fluidic port 28-7 throughflow-through conduit 28-11 and out fluidic port 28-1 to the microreactor12.

As demonstrated in FIG. 4, diluted sample flows from the mixing tee 18to the microreactor 12, as illustrated by arrows. The microreactor 12 isset to operate at the proper temperature, pressure and time for sampleto react with the reagent or other reactants, and to permit the reactionto go to completion. Essentially, diluted or un-diluted sample andreagent are provided to the microreactor 12 at a specific rate to ensurethat proper ratios of both are maintained. Sample and/or reagent can bedischarged through the injection valve 30 via the second sample loop 42to the waste reservoir 38.

Furthermore, during sample preparation, the sample pump 32, the diluentpump 34 and the reagent pump 36 are preferably turned on. Alternatively,during sample preparation, either sample pump 32 or the diluent pump 34can be turned off provided the other remains on. For example, if thesample pump 32 is turned off, the configuration of the priming valve 24is changed by rotating clockwise one position and external samplingvalve 22 is changed by rotating one position either counterclockwise orclockwise. On the other hand, if the dilute pump 34 is turned off andthe sample pump 32 remains on, the configuration of the dilute valve 26changes by rotating the clockwise one position.

Microreactors

Microreactors are small scale, continuous flow reactors. Microreactorsare also sometimes referred to as microstructured devices ormicrochannel devices. In the microreactor, chemical reactions go forwardin a confined environment and over wide ranges of temperature andpressure, each of which can be optimized according to the user'spreferences to ensure completion of the specified chemical reaction.Microreactors can be manufactured from a range of materials thatinclude, but are not limited to, glass, silicon-glass, ceramic,stainless steel or polymers.

There are different types of microreactors including, but are notlimited to, chip, capillary, microstructured and industrialmicroreactors. The microchannel design includes a mesh, catalyst-trap,micro-packed bed, falling film, and/or meandering channels.Microreactors can include mixing units, flow distributors, multiplechannels, and means for immobilizing catalyst particles. They can alsohave a variety of channel geometries, diverse mixing and heat exchangestructures. See, e.g., Jensen K. F., Reizman B. J., Newman S. G., Toolsfor Chemical Synthesis in Microsystems, May, 2014 at pp. 1-2. Forexample, the mixing units, or micromixers, made of glass withvariability in outlet geometries, such as triangular-shaped,heart-shaped, linear and so on, allow the visual investigation of themixing process and the generation of emulsions.

Generally, microreactors can have channels with diameter ranges betweensub-mm to tens of mm range and can have surface to volume ratios fromabout 1,000 to more than about 50,000 m²/m³. For example, the gas-liquidtypes of microreactors include, but are not limited to, the followingexamples of microreactors: micro bubble column microreactors (1100μm×170 μm channel size) have an interface area of about 5,100 m²/m³,micro bubble column microreactors (300 μm×100 μm channel size) have aninterface area of about 9,800 m²/m³, micro bubble column microreactors(50 μm×50 μm channel size) have an interface area of about 14,800 m²/m³and falling film microreactors (300 μm×100 μm channel size) have aninterface area of about 27,000 m²/m³.

In a microreactor, sample and reagent streams are continuously pumpedinto the microreactor. Reactants can be mixed and reacted in themicroreactor. The reaction product can leave the microreactor as acontinuous stream. Walls of microchannels typically have high heattransfer coefficient of at least about 1 MW per m³ per K, enabling heatremoval more efficiently and allowing critical reactions to be performedsafely at high temperatures, such as for example nitration reactions.Flow capabilities of microreactors range from about 0.45 ml to about 260ml per single plate per minute and throughput of microreactors can rangefrom about 2 g/min to about 4500 g/min, or up to hundreds of kg/hr. Forexample, in the liquid-liquid capillary microreactors with slug, bubbly,parallel and annular flow hydrodynamics, the flow rate is directlydependent on capillary length and aqueous-to-organic volumetric flowratio. See, e.g, van Duijn, C. J. Liquid-Liquid Microreactors for PhaseTransfer Catalysis, December, 2011, Chapter 2, pp 25-26. Additionally,certain industrial microreactors can operate at high pressures of up to600 bar in stainless steel microreactors.

Microreactors are used for a variety of chemical reactions and can beused in many industries that include but not limited to pharmaceutical,chemical, petrochemical and petroleum fields. The most common types ofderivatization include the addition of a chromophore or fluorescentfunctional group. Other examples of common notable reactions include butnot limited to Friedel-Crafts alkylation, ester hydrolysis, oxidations,phase transfer catalysis, emulsion polymerization, fluorination oftulene, ammonia oxidation, aromatic nitration, ethane epoxidation, anddehydration of methanol to form formaldehyde. For instance, nitration ofaromates with dinitrogen pentoxide is a liquid phase reaction with afast reaction time of less than 10 seconds and must be performed attemperatures below 50° C., requiring extensive cooling capabilities. Inaddition, this reaction is highly exothermic (−500 kJ/mol). Generally,sample components with active functional such as, but not limited to,alcohols, phenolic, amine, carboxyl, olefin, and others, are candidatesfor derivatization.

Furthermore, microreactors can be used for derivatization chemistry foranalysis of a broad range of samples that contain a suitable activefunctional group (or groups) available for derivatization (or chemicalreaction) such as polar groups (amines). Derivatization includes achemical reaction between an analyte and a reagent to change thechemical and physical properties of the analyte. Advantages ofderivatization include improved detectability, change in molecularstructure of analyte for better chromatography, increase volatility,change the matrix for better separation and stabilization an analyte.Ideally, derivatization reactions should be rapid, quantitative andproduce minimal by-products. Excess reagent should not interfere withthe analysis can should be easily removed.

For example, Waters AccQTag method is a precolumn derivatizationtechnique for peptide and protein hydrolysate amino acids. The AccQTagAmino Acid Analysis method can utilize a derivatizing reagent, such asAccQFluor reagent 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate(“AQC”), which forms stable derivatives with primary and secondary aminoacids in a matter of seconds, with the microreactor providing propertemperature and pressure conditions. See, e.g., EP 0 533 200 B1 atparagraphs [0011], [0020], [0021], [0023], [0026] and [0034],incorporated by reference as well as the entire contents and teachingsof which are incorporated herein by reference. The amino acidderivatives can then be injected directly into the column withoutfurther preparation. The amino acid derivatives can then be injecteddirectly into the column without further preparation.

Other useful reagents include, but are not limited to, molecules used torapidly label molecules in a sample in order to improve detectabilityfor subsequent analysis by liquid chromatography, mass spectrometry,fluorescence and ultra-violet detection. See, e.g., WO2013/049622 atpage 2, lines 11 through page 3, line 20; page 9, line 14 through page13, line 2; page 22, line 23 through page 30, line 20; and page 32, line15, incorporated by reference as well as the entire contents andteachings of which are incorporated herein by reference. Here, the MSactive, fluorescent molecules are useful as reagents for rapid taggingof glycans such as N-linked glycans and other bio-molecules such asproteins and peptides and amino acids. These MS active, fluorescentmolecules can have three functional components: (a) a tertiary aminogroup or other MS active atom; (b) a highly fluorescent moiety, and (c)a functional group that rapidly reacts with amines, such as anisocynanate or succidimidylcarbamate. The reactive functional groupprovides rapid tagging of desired bio-molecules, and the fluorescentmoiety provides for a strong fluorescent signal. The tertiary aminogroup substituent provides a strong MS signal. In another aspect, theserapid tagging MS active compounds do not have to have a fluorescentmoiety.

There are many advantages to using microreactors as most chemicalreactions generally benefit from a continuous process where solids arenot present. For example, microreactors provide high surface-to-volumeratio which allows for enhanced mass and heat transfer and eliminatesreaction hot spots. Rapid heat transfer allows increased contact betweenmolecules of the reactants, such as between the sample and thederivatization reagent, for a sufficient enough time to allow a reactionto go to completion. In other words, due to small surface-to-volumeratios that enable microreactors to absorb heat created from a reactionmuch more efficiently. Local temperature gradients that affect reactionrates are much smaller in the microreactors which allows for betterkinetic investigations. In addition, heating and cooling a microreactoris much quicker than traditional vessels used for chemical reactions,allowing very low operating costs. The superior heat exchange, heattransfer and cooling capabilities are maintained by sandwiching a thinreaction layer between cooling plates and increasing lateral size whilekeeping a constant reactor channel depth.

In addition, microreactors provide laminar flow conditions, uniformresidence time of the sample and reagent molecules, have high-throughputwith minimal amounts of sample used, low manufacturing, operating andmaintenance costs. Furthermore, when a larger amount of product isdesired, the use of microreactors allows for seamless transfer offavorable mass and heat transfer conditions established on a smallerresearch scale to industrial scaled-up manufacturing. Thus, scale-up istypically achieved by increasing reactor size while preserving heat andmass transfer advantages, and then multiplying up the resulting smallernumber of larger reactors.

Microreactors are available for small-scale and commercial use.Microreactors range from smaller devices with lateral dimensions belowabout 1 mm to scaled-up commercial microreactors for industrial chemicalproduction set-ups. For seamless scale-up, modular microreaction systemsare usually employed. Microreactors can include a single module capableof supporting mixing, heat exchange and reaction simultaneously.Alternatively, microreactors can include one or more mixing modules,heat exchange modules and/or reaction modules. Generally, at the pointof scale-up, a module with more channels is employed assuring identicalprocess results between the pilot, or discovery process and theproduction stage, as the physical condition in each channel remains thesame.

For example, during the discovery process, results can be measured in μgto mg. With an automated flow chemistry platform for rapid reactionscreening, the type of microreactor that can be employed could have a300 μm channel width such as in the Labtrix® System, for examplecurrently sold by Chemtrix. Such systems can include scouting andoptimization, kinetic data generation, process feasibility studies,process validation and additive screening. These systems can performsyntheses at temperatures between about −20° C. to about 195° C. and atpressures up to about 25 bar. The system can evaluate many reactionparameters in a short period of time with very little raw material.

For the development stage, modular scalable flow reactor systems such asthe KiloFlow® system currently sold by Chemtrix can be used to supportreaction development up to Phase 3 within a standard fume hood andwithout re-optimization or change in mixing efficiency. These systemscan perform syntheses at temperatures between about −15 and about 150°C. having reaction times in the range of about 1.0 second to about 100minutes. Production capabilities range between about about 10 to about6000 milliliters per hour. Each module can have static mixers in therange of about 60 to about 250 ms and a thermal regulator which enablesthe use of process parameters in the discovery stage to provide the sameresults on a gram to kilogram scale where a single channel or parallelchannel reactor are used. Furthermore, syringe pumps can be selected forhigh precision dosing of reagent solutions at high pressure (maximumoperating pressure at about 20 bar). To allow for continuous dosing ofreagents to the reactor, the pumps can be operated as dual syringepumps, for example, with four pump units used to deliver two reagentsolutions in the system. Using software, synchronized control over alldosing units can be achieved to allow access of flow rates of about 0.1to about 50 min—1 per reagent feed (using 25 ml syringes) for example.Single dosing is also possible over the range about 0.01 to about 50milliliters (ml)—1 using 25 ml syringes. In addition, reagents can bepre-heated before transferring to a reactor module where they are mixed.The heat exchangers can be thermally regulated using a closedrecirculating thermostat. Within the system, heat exchange modulessometimes have two functions: (1) to thermostat reagent feeds and (2)bring reaction product stream to ambient temperature ahead of samplecollection.

For production, industrial flow reactors can execute hundreds ofreactions per day and allow high production volumes in flow, producingat the ton-scale for the execution of highly profitable processes. Forexample, Plantrix® industrial flow reactor currently sold by Chemtrix isa modular reactor that allows high production volumes in flow and canproduce at the ton-scale. Because this type of reactor is a modularreactor, there is less scale-up risk associated with its use. Thedifferent reactor modules can be installed, enabling the system to betailored to a specific process by varying mixing time, residence timeand quench. Plantrix® reactors can handle volumes from about 2.9 ml toabout 5 L per reactor system and have temperature resistance up to about1500° C., with thermal conductivity five times higher than stainlesssteel. In addition, using millimeter channel dimensions, these reactorscan tolerate the use of solids in the process stream up to about 100 μmin size. Furthermore, Plantrix® reactors can be used for a wide range ofchemical process including very challenging reactions, such as, but notlimited to, fast exothermic reactions, reactions with aggressive media,reactions employing unstable intermediates, hazardous reactions, and aresuitable for both acidic and alkaline materials. Sample Loading

FIG. 5 shows the automated sampling and reaction system 2 having valvesconfigured to load sample into the second sample loop 42 of theinjection valve 30. The configuration of the external sampling valve 22,the priming valve 24, the diluent valve 26 and the reagent valve 28remain as shown in FIG. 4. However, the configuration of the injectionvalve 30 is changed to a second configuration. The injection valve 30 isrotated clockwise by one port position. Rotation of the injection valve30 counterclockwise by one port position achieves the sameconfiguration. The sample pump 32, the diluent pump 34 and the reagentpump 36 are turned to discharge sample into second sample loop 42 of theinjection valve 30, as illustrated by arrows. Specifically, in thisconfiguration, flow-through conduit 30-12 of the injection valve 30provides a fluidic pathway from the microreactor 12 into fluidic port30-3 of the injection valve 30 and out fluidic port 30-4 through thesecond sample loop 42 into fluidic port 30-1 through flow-throughconduit 30-12 and out fluidic port 30-2 to the waste reservoir 38.

Sample Injection

FIG. 6 shows the automated sampling and reaction system 2 having valvesconfigured to inject sample into the column 6. The configurations of theexternal sampling valve 22, the priming valve 24, the diluent valve 26,the reagent valve 28 and the injection valve 30 change from the loadingstep immediately described above.

To introduce the diluted and derivatized sample to the solventcomposition stream, the injection valve 30 is rotated counterclockwiseby one port position back to a first configuration such that theflow-through conduit 30-11 connects fluidic port 30-1 to fluidic port30-6 to provide a fluidic pathway from the second sample loop to thecolumn 6 or detector (not shown). In addition, the flow-through conduit30-13 connects fluidic port 30-4 to fluidic port 30-5 to provide afluidic pathway from the solvent delivery system 8 to the second sampleloop 42. Clockwise rotation of the valve 30 by one port position willachieve the same configuration. Sample contained therein in the secondsample loop 42 is introduced into solvent composition stream arrivingfrom the solvent delivery system 8.

In addition, the external sampling valve 22, the priming valve 24, thediluent valve 26 and the reagent valve 28 can optionally remain in theconfiguration as shown in FIG. 5 but preferably change to configurationsin the collection step and as shown in FIG. 2. More specifically, theexternal sampling valve 22 is rotated counterclockwise by one portposition (clockwise rotation by one port position achieves the sameconfiguration). As described above, this configuration provides afluidic pathway between the reactor 10 and the collection reservoir 44via the external sampling valve 22 through the first sample loop 40.

Similarly, the priming valve 24, the diluent valve 26 and the reagentvalve 28 can optionally remain in the configurations as shown in FIG. 5but preferably are changed to the configurations as shown in FIG. 2.Each of the valves rotates independent of the other clockwise by oneport position. In addition, the sample pump 32, the diluent pump 34 andthe reagent pump 36 can be turned off during the injection step.However, for speed of sampling and preparation, the sample pump 32, thediluent pump 34 and the reagent pump 36 remain on to be refilled andready for the next injection as shown in FIG. 2.

Example 1 Chemistry of AQC Derivatization

AccQTag derivatization reaction is appropriate for the systems andmethods described herein. For example, AccQTag derivatization has beenin use for over 10 years. See e.g., EP0533200 B1 at paragraphs [0011],[0020], [0021], [0023], [0026] and [0034], incorporated herein byreference. Here, a reagent reacts with non-protonated primary andsecondary amino acids in a largely aqueous environment to form productsthat are readily detected by a UV detector. Since the same group isbeing added to each of the amino acids, the extinction coefficients ofthe derivatized amino acids are very much the same. In other words, theresponses for equimolar amounts of the amino acids are very similar. Thecomplete chemistry occurring in the reaction tube is shown immediatelybelow.

As shown, the primary and secondary amino acids undergo reaction withthe reagent on a time scale of tens of milliseconds and is thereforecomplete in less than about 1 second. The excess reagent then reactswith water on a time scale of 10s of seconds and forms byproducts thatdo not interfere with the amino acid analysis, for example. No specialhandling is required to remove the excess reagent and the reaction maybe carried out on the bench top.

Example 2 Manual Derivatization of Amino Acids Using AccQTag Kit

Unlike systems and methods described herein, the following scheme isexemplary of steps taken in manual derivatization of sample containingamino acids. This methodology is presented in contrast to the automatedsampling and reaction system and associated methods described herein.First, reagent is reconstituted using 3×1 mL rinse of pipette tip withdiluent. The reagent vial is tapped on the table to assure material isat the bottom of the vial. 1 mL of reagent diluent is transferred to thereagent vial and vortex the vial and placed atop a heatblock set to 55 Cfor ten minutes. As provided in Table 1, borate buffer is then added toblanks, standard sample and sample and any necessary base volumes toWaters Total Recovery Vial and vortex to mix thoroughly. For example, 20μL of reconstituted reagent is added below liquid level in TRV to eachvial, then capped and vortex immediately. The vial is allowed to standat room temperature for one minute, then placed in 55° C. heat block for10 minutes. As described herein, these steps can now be performed in themicroreactor and automatically.

TABLE 1 Derivatization Standard, Volumes (μL) Gradient Blank (Reagent)Blank Sample AccQ•Tag Ultra Borate 80 80 *70  Buffer AccQ•Tag UltraReagent 20 Diluent Standard or Sample 10 NaOH for neutralizing * samplereconstituted AccQ•Tag 20 20 Ultra Reagent

Some guidelines should be followed for successful derivatization. First,the amount of sample present needs to be within the dynamic linear rangeof the method and above likely environmental contaminant levels (>1picomole on column for least abundant amino acid (LAAA) and <140 nanomolof total amines in the derivatization cocktail. Second, pH should bebetween 8 and 10, to assure amines are unprotonated. Borate volume inthe derivatization needs to be sufficient to neutralize 0.1 N acid. Forsamples with more than 0.1N acid, adjust with equal volume of base atthe same concentration. The pH must not be so low or so high that thereagent is destroyed. Third, there must be sufficient excess of reagentto drive the derivatization reaction to completion. For example,˜5×molar excess of reagent over sample is required.

Other considerations for successful quantitative analysis of amino acidsinclude, first, the organic concentration of the derivatization mixturemust be high enough to keep the reagent and the derivatives in solutionbut not so high as to distort the chromatography. Second, the amino acidconcentration must be above the required sensitivity limits. Third, thesample must not be contaminated with amino acids, with proteins, or withother environmental amines.

Furthermore, data should be reviewed to confirm that there are nosignificant peaks in the gradient blank and verify that the AMQ peakheight is ≧0.9 AU for all derivatized samples. The reagent blankchromatogram should not have significant amino acid contamination (≦50femtomoles, Gly ≦100 femtomoles) and multiple injections of the standardshould overlay well. Mono-derivatized lysine should not exceed 2% of Pheheight. Also, retention time alignment of amino acid peaks should becompared with component markers and adjusted.

To troubleshoot poor quantitation, one should consider that hydrophobicamino acids come out of solution if not enough organics are present. Ifthere are too many organics in derivatization, this will distort earlyeluting peaks. Furthermore, evaporation of organic in the secondarysample can also result disproportionately larger early peaks and smallerlater peaks. As to derivatization-related quantification, the AMQ peaksize should be ≧0.90 AU. Furthermore, Asp, Glu, Lys, Ala are mostsensitive to pH and not enough excess reagent. Moreover, Ala/Phe ratioof the standard can indicate if the reagent is bad or if there is aproblem with the Borate buffer and the presence of mono-derivatizedLysine indicates incomplete derivatization. Also, certain quantitationproblems are hydrolysis-related including: (1) methionine recoveryvaries with oxygen exposure; (2) tyrosine, threonine, and serine aregradually destroyed; (3) hydrophobics may be released slowly; (4) toomuch solids or not enough acid slows or stops hydrolysis; and (5)protein or amino acid contamination introduced.

Therefore, for troubleshooting, identifying obvious issues such asinstrumentation, chemistry or sampling problems is key and using fresh,tested columns and bottled, tested eluents is essential. To judge theresults, retention times must be the same and correct, peak areas mustbe the same and must match results acquired on installation and oneshould compare area ratios with installation result.

One of ordinary skill in the art will appreciate further features andadvantages of the invention based on the above-described embodiments.Accordingly, the invention is not to be limited by what has beenparticularly shown and described, except as indicated by the appendedclaims. All publications and references cited herein are expresslyincorporated herein by reference in their entirety.

We claim:
 1. An automated sampling and reaction system comprising: anexternal sampling valve connected to a priming valve wherein theexternal sampling valve is configured to draw sample from a reactor or areactor stream and the priming valve is configured to discharge wash tothe external sampling valve; a microreactor in fluidic communicationwith the external sampling valve and connected to a reagent valve,wherein the reagent valve is configured to discharge reagent to themicroreactor and the external sampling valve is configured to dischargesample to the microreactor to form a secondary sample; and an injectionvalve connected to the microreactor and in fluidic communication with acolumn or detector, wherein the injection valve is configured todischarge the secondary sample into a solvent composition stream.
 2. Theautomated sampling and reaction system of claim 1, further comprising amixing tee connected to the external sampling valve and the microrector.3. The automated sampling and reaction system of claim 2, furthercomprising a diluent valve connected to the mixing tee, wherein thediluent valve is configured to discharge diluent to the mixing tee. 4.The automated sampling and reaction system of claim 1, furthercomprising a pumping system having one or more pumps working incombination with the priming valve and the reagent valve, wherein thepriming valve is configured to draw wash from a wash reservoir ordischarge wash to the external sampling valve and the reagent valve isconfigured to draw reagent from a reagent reservoir or to dischargereagent into the microreactor.
 5. The automated sampling and reactionsystem of claim 3, further comprising a pumping system having one ormore pumps working in combination with the priming valve, the diluentvalve, and/or the reagent valve wherein the priming valve is configuredto draw wash from a wash reservoir or discharge wash to the externalsampling valve, the reagent valve is configured to draw reagent from areagent reservoir or to discharge reagent into the microreactor, and thediluent valve is configured to draw diluent from the diluent reservoirand discharge diluent to the mixing tee.
 6. The automated sampling andreaction system of claim 1, wherein in a first configuration, theexternal sampling valve draws a discrete amount of sample or continuoussample from a reactor or reactor flow stream and, in a secondconfiguration, the external sampling valve discharges drawn sample via abackwash discharged from the sample pump to the mixing tee.
 7. Theautomated sampling and reaction system of claim 3, wherein the diluentvalve is configured to draw diluent from a diluent reservoir.
 8. Theautomated sampling and reaction system of claim 1, wherein the systemoperates under a pressure greater than about 1 atmosphere.
 9. Theautomated sampling and reaction system of claim 1, wherein the system isconfigured to draw from a non-pressurized source and further comprisesat least one external auxiliary sampling valve and at least one externalsample pump wherein the external auxiliary sample valve is connected tothe external sample pump, the external sampling valve and to the reactoror reactor stream.
 10. The automated sampling and reaction system ofclaim 9 wherein the system further comprises at least one selectionvalve connected to the external auxiliary sampling valve and to theexternal sampling valve.
 11. The automated sample and reaction system ofclaim 1 wherein the system comprises a plurality of the externalsampling valves and a selection valve connected to each of the externalsampling valves.
 12. The automated sample and reaction system of claim 1wherein the microreactor is a chip, capillary, micro-structured orindustrial type microreactor.
 13. A liquid chromatography systemcomprising the automated sampling and reaction system of claim
 1. 14. Amethod of quantitative analysis of a liquid solution comprising thesteps of: selecting a source of sample from a reactor or a reactor flowstream; acquiring sample from the reactor or the reactor flow streamthrough an external sampling valve, wherein the external sampling valveis configured to draw sample into a first sample loop; drawing washthrough a priming valve in fluidic communication with the externalsampling valve wherein the priming valve is configured to discharge washfrom the second sample loop to the external sampling valve; reactingsample with a reagent discharged from a reagent valve into amicroreactor wherein the microreactor is in fluidic communication withthe external sampling valve and the reagent valve is connected to themicroreactor to form a secondary sample; discharging the secondarysample into a second sample loop of an injection valve; and injectingsample from the injection valve into a solvent composition stream influidic communication with a column or detector.
 15. The method ofquantitative analysis of a liquid solution of claim 14, wherein thesample is acquired from the reactor or the reactor flow stream operatingat pressure of more than one atmosphere.
 16. The method of quantitativeanalysis of a liquid solution of claim 14, wherein sample is acquiredfrom the reactor or the reactor flow stream operating at a pressure ofone atmosphere or less.
 17. The method of quantitative analysis of aliquid solution of claim 14 wherein the reagent is an MS active,fluorescent rapid tagging reagent.
 18. The method of quantitativeanalysis of a liquid solution of claim 14, wherein the secondary sampleis an MS active, fluorescent biomolecule.
 19. The method of quantitativeanalysis of a liquid solution of claim 16, wherein sample is acquired byan external sample pump connected to an external auxiliary samplingvalve, the external pump discharges drawn sample via a backwash from asample pump to a mixing tee connected to the microreactor.
 20. Themethod of quantitative analysis of a liquid solution of claim 19 furthercomprising the step of quenching sample at the mixing tee.